No abstract

ObjectivesExperimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a "skin equivalent" and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells.MethodsPrimary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry.ResultsDifferent protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.Conclusion3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue.

In human cholesteatomas, Cx43 was expressed in whole suprabasal layers, except in the basal layer, and Cx26 was usually expressed in the suprabasal and basal layers. However, normal RASs showed weak expression of Cx43 in the upper spinosal and granular layers (with no expression in the basal layers) and restricted localization of Cx26 in the basal layer. The expression of Cx43 and Cx26 in ECSs was weak but showed similar patterns to that of cholesteatoma. RT-PCR and Western blotting showed that the expression of Cx43 and Cx26 was higher in cholesteatoma than in RASs. Epithelial cells expressing Cx43 and Cx26 in cholesteatoma were not exactly identical to Ki67-expressing cells on immunohistochemical staining.

No abstract

The purpose of this study was to establish the eventual presence of progesterone receptor (PGR) and oestrogen receptor (EGR) in human middle-ear cholesteatoma (MECh) tissues and to compare their expression between male and female patients. An immunohistochemical technique was employed for detection of PGR- and EGR-specific immunoreactivity in MECh samples using formalin-fixed paraffin-embedded tissue sections. The positive results were verified with reverse transcriptase polymerase chain reaction (RT-PCR). The morphological study revealed stable expression of PGR in suprabasal layers of all cholesteatoma samples. Weaker immunoreactivity for PGR was demonstrated in external auditory canal skin (EACS) samples in comparison with MECh, while PGR-specific staining was not observed in retroauricular skin (RAS) samples. EGR was detected only at mRNA levels. Stronger expression of EGR PCR products was disclosed in female cholesteatoma samples, while PGR mRNA was predominantly detected in male cholesteatoma specimens. Our preliminary experimental results give us ground to assume that female sex hormones may stimulate proliferation and affect differentiation of MECh keratinocytes.

No abstract